[unreadable] GenWay Biotech, Inc., a protein and antibody solutions provider, is proposing to apply its technology in a collaborative effort with the Deisseroth laboratory at the Sidney Kimmel Cancer Center to develop a method to break the immune tolerance of tumor associated antigens (TAA) on cancer cells. The Deisseroth laboratory showed that the subcutaneous injection of the Ad-sig-TAA/ecdCD40L adenoviral vector results in the in vivo activation and loading of dendritic cells (DCs) with TAA. The vector is engineered so that a fusion protein (TAA/ecdCD40L) consisting of a TAA linked to the extracellular domain (ecd) of the CD40 ligand (CD40L) is secreted from vector infected cells for 10 days in the vicinity of the vector injection site. The activated and TAA loaded DCs then migrate to regional nodal tissue. Two TAA were studied: the HPV E7 foreign antigen and the human MUC-1 (hMCU-1) self antigen. The Deisseroth laboratory showed in a mouse model that the Ad-sig-TAA/ecdCD40L vector injection induced an immune resistance to the growth of TAA positive tumor ceils for up to a year. This immunity could be transferred from vaccinated donor mice to unvaccinated mice through CD8 T cell lymphocytes. We are proposing to study the boosting effect of hMUC-1/ecdCD40L protein injections on an immunization primed with the Ad-sig-ecdhMUC-1/ecdCD40L vector. We will first test for differences in the boosting effect of subcutaneous injections of the ecdhMUC-1/ecdCD40L protein prepared from either bacterial or mammalian cell expression systems. This study will have the following specific aims (SA): SA#1: To test for any differences between E coli vs 293 cell-derived ecdhMUC- 1/ecdCD40L proteins in boosting an ecdhMUC-1 specific CTL response. SA#2: To assess in vivo the boosting effect of the ecdhMUC-1/ecdCD40L protein on preventing the growth of hMUC-1 positive mouse tumor cells in hMUC-1 .Tg transgenic mice primed with the Ad-sig-ecdhMUC-1/ecdCD40L vector. SA#3: To investigate in vivo the boosting effect of the ecdhMUC-1/ecdCD40L protein on suppressing pre-established hMUC-1 positive syngeneic mouse tumor cell growth in hMUC-1.Tg transgenic mice primed with the Adsig- ecdhMUC-1/ecdCD40L vector. These specific aims are designed to lead to a Phase II SBIR application to support the development of a clinical trial of the TAA/ecdCD40L vaccine therapy which could be important for immunotherapy of the many forms of epithelial malignancy in which the hMUC-1 antigen is overexpressed. [unreadable] [unreadable] [unreadable]